基因表达与mRNA结构的关系

基因的转录调控和转录后水平的调控在基因表达过程中起着重要作用。mRNA的结构与基因表达调控的关系非常密切。目前对于mRNA结构对表达的影响因素,主要集中于起始密码子和S-D序列的结构和间隔长度、基因和基因间的间隔区序列和长度,5’末端与3’末端非翻译区、多聚（A）尾、内含子序列对翻译起始效率、发夹结构对mRNA的稳定性的影响和mRNA翻译起始区等对基因表达影响。     

大肠杆菌、酵母和果蝇基因保守位点的信息熵分析

对大量的大肠杆菌（Escherichia coli)、酵母（Yeast)和果蝇（Drosophila melanogaster）已知基因起始密码子和终止密码子上、下游各30个碱基序列,用重新定义单碱基信息冗余（记为D1(ι）,ι是位点）和紧邻碱基的信息冗余（记为D2（ι）,统计计算每个位点的D1（ι）和D2（ι）值。从结果看,双碱基比单碱基携带更多的信息;酵母和果蝇基因起始密码子上游－3位点D1（－3）和D2（－3）有一明显峰值;大肠杆菌基因起始密码子上游SD区域D1（ι）和D2（ι）有明显峰值,与他人结论相同。发现酵母基因起始密码子下游的＋4位点与＋5位点的紧邻碱基的D2（ι）有一峰值,其关联模式为TC（联合概率为0.211)。这说明用重新定义的信息冗余去确认DNA序列中存在的保守位点是完全可行的。     

籼稻品种93-11同义密码子的使用偏性

利用籼稻品种93－11的全基因组序列及相应的EST数据,对影响同义密码子用法的若干因子进行了详细分析。指出93－11基因的表达水平（mRNA丰度）与3个同义密码子偏性指标CAI、CPP和ENC相关极显著（r=0.227^**,0.145^**和-0.147^**),表明高表达的基因其同义密码子非随机使用的程度越大;基因长度与CAI和CPP极显著负相关（r=-0.413^**和-0.480^**),与ENC极显著正相关（r=0.210^**),暗示较短的基因具有更高的转录活性;编码区G＋C含量对其同义密码子偏性的贡献率远高于mRNA丰度和基因长度,G＋C含量与CAI、CPP和ENC相关系数分别高达0.877^**,0.832^**和-0.740^**;起始编码区内A、T、C、G4种碱基呈明显的3周期振荡,尤以ATG下游第一个密码子所在的3个位点（＋4、＋5和＋6）偏置最强烈,由此认为在这3个特殊位点有较高的自然选择压存在;93－11中25个最优密码子的首次确定将对水稻转基因具有指导意义。     

mRNA翻译起始区二级结构改变对干扰素基因在大肠杆菌中翻译的影响

为研究mRNA翻译起始区结构与基因表达的关系,利用密码子的简并性,在不改变表达产物氨基酸序列的前提下定点突变α8干扰素及αA干扰素衍生物基因的5′端若干位点,使其与表达载体重组后转录形成的mRNA翻译起始区结构发生改变。SDS-PAGE及活性测定证实这些改变提高了外源基因的表达水平。RNA斑点印迹表明突变前后基因转录水平差别不大,表达水平的提高主要由于翻译效率的提高。mRNA翻译起始区二级结构预测提示其生成自由能(ΔG)的变化可能与表达水平的提高有关。     

影响链球菌属肺炎球菌基因组密码子使用的因素分析

链球菌属肺炎球菌（Steptococcus pneumoniae)的完整基因组序列已经测定完毕并于近期发表,对肺炎球菌基因组序列进行了详细分析,研究了基因组密码子的使用模式和影响密码子使用的因素,高水平高达基因的密码子第三位碱基使用胞嘧啶（C）的频率比表达水平低的基因使用C有显著的提高,表达水平较低的基因在密码子的第三位碱基更趋向使用嘌呤）,基因的表达水平与对应分析的第一条向量轴呈显著相关（R＝0．86）,比较表达水平高,低的两组基因的密码子使用模式发现,基因的表达水平对于密码子使用有显著的影响,基因碱基G＋C的组成与基因的表达水平（R＝0．44）,对应分析的第一条向量轴（R＝0．5）有显著的相关,对基因的表达水平,密码子的使用有显著的影响,通过GC－skew,蛋白质的疏水性,基因的长度分析,发现不同长度的基因表达水平,GC含量,GC3s有差异,结果表明,在表达水平上的自然选择以及基因的碱基组成是影响肺炎球菌基因密码子使用的主要因素,基因的长度对密码子的使用有一定影响。     

大肠杆菌精氨酰tRNA合成酶基因（argS）的上游非编码区存在一个负调控区

含大肠杆菌精氨酰tRNA合成酶（ArgRS）基因(argS）的pUC18重组质粒,在大肠杆菌TG1转化子中能够高表达ArgRS近1000倍。为了研究大肠杆菌argS的表达调控,构建了一系列的缺失突变。分别缺失全部上游序列（argS△1）、Shine-Dalgarno（SD）区（argS△2）和缺失启动子－10区下游（相当于翻译起始位点－65nt,argS△3）前的上游序列后的变种argS都不能表达ArgRS。而缺失－122nt（距翻译起始位点－180nt,argS△8）、－70nt（距翻译起始位点－128nt,argS△7）、－52nt（距翻译起始位点－110nt,argS△6）、－35区9距翻译起始位点－94nt,argS△5）、启动子－10区（距翻译起始位点－71nt,argS△4）前的上游序列后,这些缺失突变基因的表达水平与野生型argS接近。但argS△4、argS△5、argS△6都会形成部分包涵体。通过RNA斑点杂交测定发现,argS△4、argS△5和argS△6的mRNA转录量为argS及argS△7的2到3倍。即－52nt和－70nt之间的19个碱基（AATAGTGAAAACGGCAATA)可能是大肠杆菌argS舞场康负调控区。该元件的缺失将使得ArgRS过快表达并导致部分蛋白质形成包涵体。凝胶阻滞分析也发现在细胞粗抽液中有一个因子可以专一地与这个负调控元件结合,它可能参与该基因的表达调控。精氨酸专一性地诱导argS的转录,其作用与上述转录负调控区相关。     

敏捷气热菌起始密码子侧翼序列与密码子偏好及基因长度相关性研究

好氧超嗜热古菌敏捷气热菌 (Aeropyrumpernix)mRNA中起始密码子AUG侧翼序列的保守性以及它与密码子使用偏好及基因长度之间具有相关性。AUG侧翼序列的保守性由M1(1)值表示 ,AUG侧翼序列对翻译起始的有效性由AUGCAI值表示。研究表明 :高表达和低表达基因的 - 2 0位到 13位中某些位点的保守性存在差异 ,其中高表达基因的 - 4位和 - 3位可能与其高表达的特性有关 ;在A .pernix中一个普遍的趋势是 :较短的基因有较高的表达效率 ,较长的基因的表达效率较低。与仅使用密码子偏好相比 ,将AUGCAI值引入到研究古菌在翻译水平上的自然选择更准确、更具有广泛适应性     

寿光鸡ADSL基因5′调控区的克隆、序列分析以及单核苷酸多态性的研究

腺苷酸琥珀酸裂解酶(ADSL)是双功能酶,催化嘌呤核苷酸的起始合成与嘌呤核苷酸的循环。通过对寿光鸡ADSL 5′调控区1035bp的序列进行克隆和测序分析,发现其具有典型的管家基因特征：没有真核基因明显的TATA盒和CAAT盒出现,并且位于起始密码子(ATG)前234个碱基具有非常高的GC含量达72．65％。在邻近起始密码子“ATG”处,5′调控区含有两个核呼吸因子-2(NRF-2),在相同位置上人类ADSL基因也具有与此类似的两个核呼吸因子-2结合位点,被认为在嘌呤核苷酸合成途径起到重要作用。值得一提的是位于寿光鸡5′侧翼调控区-27号碱基发生C→T突变,存在于所有研究的寿光鸡个体中,频率为1。该突变使得本来不是NRF-2(核呼吸因子2)结合位点的CTCC突变为NRF-2结合位点CTTC。而人类却恰恰与此相反第一个NRF-2结合位点发生了突变(CTTC→CTCC),并导致出现ADSL缺陷症状。     

短小芽孢杆菌碱性蛋白酶基因启动子的克隆、鉴定及其应用

利用TAIL-PCR(Thermal asymmetric interlaced PCR)从短小芽孢杆菌基因组中扩增到碱性蛋白酶基因编码区上游的启动子片段。对该片段的序列测定和分析表明, 此片段长797 bp, 但与基因表达有关的序列长约390 bp。对启动子片段进行不同长度的缺失突变, 以获得最小的基因启动子片段, 结果表明, 该基因起始密码子上游约160 bp的DNA片段就可以启动基因的表达。将含有该片段的碱性蛋白酶基因WApQ3插入大肠杆菌-芽孢杆菌穿梭质粒载体pSUGV4中, 构建了碱性蛋白酶基因表达质粒pSUBpWApQ3。将该质粒分别转入枯草芽孢杆菌和短小芽孢杆菌中表达, 可在胞外检测到碱性蛋白酶活性, 最高酶活分别为466.5 U/mL和3060 U/mL。     

水稻OsGSTL1启动子的分离和表达分析

从水稻基因组文库中筛选得到一个水稻GST基因,命名为OsGSTL1.半定量RT-PCR分析表明OsGSTL1基因的表达不受绿磺隆、乙烯利、脱落酸、水杨酸和茉莉酸甲酯的诱导,因此该基因可能与植物抗逆性无关.为了研究OsGSTL1启动子在植物体内的表达特性,将OsGSTL1起始位点5＇端上游不同长度的调控序列与报告基因GUS融合,并在洋葱表皮瞬间表达和拟南芥中稳定表达.研究表明：在洋葱表皮细胞中,160bp及更长的上游调控序列均能启动GUS基因的表达;而在转基因拟南芥中,含有2155 bp的上游序列的PGZ2.1：：GUS具有时空表达的特性,在转基因的早期幼苗中GUS基因在子叶中特异性表达,但在根中没有表达;而在幼苗生长的后期,根、茎、叶中都有少量的表达.但包含1 224 bp的上游序列的PGZ1.2：：GUS却表现为组成型表达的特性.由此推测,OsGSTL1启动子启动的基因表达可能与幼苗的营养代谢相关;而OsGSTL1启动子的时空表达相关元件可能位于OsGSTL1翻译起始位点5＇端上游-2155 bp至-1224 bp范围内.     

Patterns of codon usage in two ciliates that reassign the genetic code: Tetrahymena thermophila and Paramecium tetraurelia

We used the recently sequenced genomes of the ciliates Tetrahymena thermophila and Paramecium tetraurelia to analyze the codon usage patterns in both organisms; we have analyzed codon usage bias, Gln codon usage, GC content and the nucleotide contexts of initiation and termination codons in Tetrahymena and Paramecium. We also studied how these trends change along the length of the genes and in a subset of highly expressed genes. Our results corroborate some of the trends previously described in Tetrahymena, but also negate some specific observations. In both genomes we found a strong bias toward codons with low GC content; however, in highly expressed genes this bias is smaller and codons ending in GC tend to be more frequent. We also found that codon bias increases along gene segments and in highly expressed genes and that the context surrounding initiation and termination codons are always AT rich. Our results also suggest differences in the efficiency of translation of the reassigned stop codons between the two species and between the reassigned codons. Finally, we discuss some of the possible causes for such translational efficiency differences.     

Codon usage in regulatory genes in Escherichia coli does not reflect selection for ''rare'' codons.

It has often been suggested that differential usage of codons recognized by rare tRNA species, i.e. "rare codons", represents an evolutionary strategy to modulate gene expression. In particular, regulatory genes are reported to have an extraordinarily high frequency of rare codons. From E. coli we have compiled codon usage data for highly expressed genes, moderately/lowly expressed genes, and regulatory genes. We have identified a clear and general trend in codon usage bias, from the very high bias seen in very highly expressed genes and attributed to selection, to a rather low bias in other genes which seems to be more influenced by mutation than by selection. There is no clear tendency for an increased frequency of rare codons in the regulatory genes, compared to a large group of other moderately/lowly expressed genes with low codon bias. From this, as well as a consideration of evolutionary rates of regulatory genes, and of experimental data on translation rates, we conclude that the pattern of synonymous codon usage in regulatory genes reflects primarily the relaxation of natural selection.     

The +4G site in Kozak consensus is not related to the efficiency of translation initiation

The optimal context for translation initiation in mammalian species is GCCRCCaugG (where R = purine and "aug" is the initiation codon), with the -3R and +4G being particularly important. The presence of +4G has been interpreted as necessary for efficient translation initiation. Accumulated experimental and bioinformatic evidence has suggested an alternative explanation based on amino acid constraint on the second codon, i.e., amino acid Ala or Gly are needed as the second amino acid in the nascent peptide for the cleavage of the initiator Met, and the consequent overuse of Ala and Gly codons (GCN and GGN) leads to the +4G consensus. I performed a critical test of these alternative hypotheses on +4G based on 34169 human protein-coding genes and published gene expression data. The result shows that the prevalence of +4G is not related to translation initiation. Among the five G-starting codons, only alanine codons (GCN), and glycine codons (GGN) to a much smaller extent, are overrepresented at the second codon, whereas the other three codons are not overrepresented. While highly expressed genes have more +4G than lowly expressed genes, the difference is caused by GCN and GGN codons at the second codon. These results are inconsistent with +4G being needed for efficient translation initiation, but consistent with the proposal of amino acid constraint hypothesis.     

Codon usage in Aspergillus nidulans.

Summary Synonymous codon usage in genes from the ascomycete (filamentous) fungus Aspergillus nidulans has been investigated. A total of 45 gene sequences has been analysed. Multivariate statistical analysis has been used to identify a single major trend among genes. At one end of this trend are lowly expressed genes, whereas at the other extreme lie genes known or expected to be highly expressed. The major trend is from nearly random codon usage (in the lowly expressed genes) to codon usage that is highly biased towards a set of 19–20 optimal codons. The G+C content of the A. nidulans genome is close to 50%, indicating little overall mutational bias, and so the codon usage of lowly expressed genes is as expected in the absence of selection pressure at silent sites. Most of the optimal codons are C- or G-ending, making highly expressed genes more G+C-rich at silent sites.     

Analysis of sequence patterns proximal to the stop codons in Oryza sativa

Abstract

A comprehensive analysis of sequence patterns around the stop codons was performed, by using more than 26,000 rice full-length cDNA sequences. Here it is shown that the bias was most outstanding at the position immediately before the stop codons (?1 codon), where the AAC codon was strongly preferred among ANC codons. Compared with other positions, the codon immediately after the stop codons (+1 codon) also displayed an apparent difference, and had a strong consensus for base A at the first, C at the second, and A at the third letters, respectively. Notably, the base biases at the positions directly downstream of the stop codons, such as the +4, +5 and +6 positions, were much stronger than other positions in the 3′-UTR region, suggesting that those base positions might act as an extended stop signal in the process of protein synthesis. Examination of the relationship between sequence pattern and gene expression level, assessed by CAI values and EST counting, revealed a tendency towards bigger base biases for highly expressed genes. It could be inferred that the translation stop signal is possibly involved in many sequence recognition elements other than the stop codons; highly expressed genes should hold strong sequence consensus around the stop codons for efficient translation termination.     

Lactic acid bacteria as prime candidates for codon optimization

In species having a strong correlation of expressivity and codon bias it has been shown that heterologous expression can be optimized by changing codons of the introduced gene towards the set of codons that the host organism naturally uses in its highly expressed genes. Even though two lactic acid bacteria are fully sequenced, there are no reports on attempts of codon optimization in the literature. In this report it is demonstrated that codons used in highly expressed genes tend to differ from the codons in lowly expressed genes, and that there is a strong correlation of codon bias and empirical expressivity (codon adaptation index) in Lactococcus lactis and Lactobacillus plantarum. This strongly suggests that codon optimization strategies could be applied to expression systems with lactic acid bacteria as producer strains. A good example of a candidate for codon optimization is the mouse interleukin-2 gene, which in its natural form has an extremely low codon adaptation index for expression in Lc. lactis.     

Studies on codon usage in Entamoeba histolytica

Codon usage bias of Entamoeba histolytica, a protozoan parasite, was investigated using the available DNA sequence data. Entamoeba histolytica having AT rich genome, is expected to have A and/or T at the third position of codons. Overall codon usage data analysis indicates that A and/or T ending codons are strongly biased in the coding region of this organism. However, multivariate statistical analysis suggests that there is a single major trend in codon usage variation among the genes. The genes which are supposed to be highly expressed are clustered at one end, while the majority of the putatively lowly expressed genes are clustered at the other end. The codon usage pattern is distinctly different in these two sets of genes. C ending codons are significantly higher in the putatively highly expressed genes suggesting that C ending codons are translationally optimal in this organism. In the putatively lowly expressed genes A and/or T ending codons are predominant, which suggests that compositional constraints are playing the major role in shaping codon usage variation among the lowly expressed genes. These results suggest that both mutational bias and translational selection are operational in the codon usage variation in this organism.     

Codon bias as a factor in regulating expression via translation rate in the human genome

We study the interrelations between tRNA gene copy numbers, gene expression levels and measures of codon bias in the human genome. First, we show that isoaccepting tRNA gene copy numbers correlate positively with expression-weighted frequencies of amino acids and codons. Using expression data of more than 14,000 human genes, we show a weak positive correlation between gene expression level and frequency of optimal codons (codons with highest tRNA gene copy number). Interestingly, contrary to non-mammalian eukaryotes, codon bias tends to be high in both highly expressed genes and lowly expressed genes. We suggest that selection may act on codon bias, not only to increase elongation rate by favoring optimal codons in highly expressed genes, but also to reduce elongation rate by favoring non-optimal codons in lowly expressed genes. We also show that the frequency of optimal codons is in positive correlation with estimates of protein biosynthetic cost, and suggest another possible action of selection on codon bias: preference of optimal codons as production cost rises, to reduce the rate of amino acid misincorporation. In the analyses of this work, we introduce a new measure of frequency of optimal codons (FOP'), which is unaffected by amino acid composition and is corrected for background nucleotide content; we also introduce a new method for computing expected codon frequencies, based on the dinucleotide composition of the introns and the non-coding regions surrounding a gene.     

Codon Usage in Plastid Genes Is Correlated with Context, Position Within the Gene, and Amino Acid Content

Highly expressed plastid genes display codon adaptation, which is defined as a bias toward a set of codons which are complementary to abundant tRNAs. This type of adaptation is similar to what is observed in highly expressed Escherichia coli genes and is probably the result of selection to increase translation efficiency. In the current work, the codon adaptation of plastid genes is studied with regard to three specific features that have been observed in E. coli and which may influence translation efficiency. These features are (1) a relatively low codon adaptation at the 5′ end of highly expressed genes, (2) an influence of neighboring codons on codon usage at a particular site (codon context), and (3) a correlation between the level of codon adaptation of a gene and its amino acid content. All three features are found in plastid genes. First, highly expressed plastid genes have a noticeable decrease in codon adaptation over the first 10–20 codons. Second, for the twofold degenerate NNY codon groups, highly expressed genes have an overall bias toward the NNC codon, but this is not observed when the 3′ neighboring base is a G. At these sites highly expressed genes are biased toward NNT instead of NNC. Third, plastid genes that have higher codon adaptations also tend to have an increased usage of amino acids with a high G + C content at the first two codon positions and GNN codons in particular. The correlation between codon adaptation and amino acid content exists separately for both cytosolic and membrane proteins and is not related to any obvious functional property. It is suggested that at certain sites selection discriminates between nonsynonymous codons based on translational, not functional, differences, with the result that the amino acid sequence of highly expressed proteins is partially influenced by selection for increased translation efficiency. Received: 21 July 1999 / Accepted: 5 November 1999